Genetic Features of Mycobacterium avium subsp. paratuberculosis Strains Circulating in the West of France Deciphered by Whole-Genome Sequencing

ABSTRACT Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis. The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis, and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

Reviewer #2 (Comments for the Author): See attachment.

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This manuscript reveals new insights into Map strains circulating in Western France. I have only one minor suggestion:
Animal 85, not 86, cited in line 244, page 11, show mixed infections as presented on table S4.

MAP WGS France Summary
Whole genome sequencing (WGS) of 200 isolates of M. avium sp. paratuberculosis (MAP) was used to analyze the genetic characteristics of MAP isolated from infected cattle in France. Results were compared with available MAP WGS findings from around the world.
Major results A closed pangenome for MAP as compared to M. avium sp. hominissuis (MAH) was confirmed with the preponderance of genes belonging to the core genome demonstrating the highly clonal nature of MAP. Only C type MAP was found, which phylogenetically distributed into 3 closely related clades. No cattle breed selectivity was apparent. Mixed infection within herds and individual animals was identified. Nonhomoplasic phylogeny based upon single-nucleotide-polymorphisms SNP alignment demonstrated the homoplasic nature of variable-number-tandem-repeat (VNTR) and short-sequence-repeat (SSR) genotyping methods. WGS thus provides resolves strain relatedness at a more granular level than traditional methods for strain identification.

Significance
The findings strongly imply that variations in MAP infection within dairy herds result primarily from herd introduction through animal trade. This is the first analysis of MAP by WGS in France, is a substantial contribution to extant databases of MAP WGS, solidifies WGS as the most reliable method of MAP strain identification, and provides a foundation for future epidemiologic investigation of this economically important disease.

Comments:
The manuscript is well written. The sequence of presentation has been carefully considered enabling the reader to work through complex methodology and analysis.
The supplemental tables and figures are all helpful and informative for readers seeking more detail.
A limitation of the study, not expressly alluded to in the discussion is that the analysis is based upon herd sampling that was intended to evaluate the dynamics of shedding in herds known to be infected. The isolates obtained are thus skewed more toward animals that are likely to be heavily infected. The relative frequency, for example, of type C clades identified may therefore not be reflective of strain virulence, but rather be a convenience sample of strain amplification within chronically infected herds. While there is no claim of epidemiological precision in the manuscript, this is an important limitation that should inform the design of future studies using WGS for epidemiologic evaluation of MAP transmission dynamic and interventional efforts to reduce herd prevalence.

Authors' response:
Thank you for the comment. There was indeed an error concerning animal 85 between the text and the table S4, thank you for pointing out this detail. This phrase L240 has been corrected and modified for clarity as follows: At the animal level the same phenomenon, i.e. clonal infection, was observed with 3 identical isolates sampled over a 2-month period from animal 85 (see Table S4, animal 85, herd 18). L242-L244 the herd number have been indicated in this version L249 animal number has been corrected, 85 to 86 in this revised version

Reviewer #2
Comments: The manuscript is well written. The sequence of presentation has been carefully considered enabling the reader to work through complex methodology and analysis. The supplemental tables and figures are all helpful and informative for readers seeking more detail. A limitation of the study, not expressly alluded to in the discussion is that the analysis is based upon herd sampling that was intended to evaluate the dynamics of shedding in herds known to be infected. The isolates obtained are thus skewed more toward animals that are likely to be heavily infected. The relative frequency, for example, of type C clades identified may therefore not be reflective of strain virulence, but rather be a convenience sample of strain amplification within chronically infected herds. While there is no claim of epidemiological precision in the manuscript, this is an important limitation that should inform the design of future studies using WGS for epidemiologic evaluation of MAP transmission dynamic and interventional efforts to reduce herd prevalence. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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